This proposal describes a genetic approach to investigating the mammalian 80S ribosome. Mutant clones that possess functionally-altered ribosomes will be isolated from a wild-type Chinese hamster lung (CHL) cell line. Selection of these mutants will involve both temperature sensitivity and resistance to drugs that affect the mammalian ribosome specifically. Mutants will be analyze by radioisotopic labelling techniques in tissue culture and protein biosynthetic assays in cell-free extracts to recognize clones whose ribosomes have been altered genetically. Ribosome subunits then will be purified to document each mutation's subunit location (40S or 60S). Structural analyses of the proteins contained by mutant and wild-type ribosome subunits will be undertaken to correlate each mutant phenotype with a particular altered protein. We anticipate that these experiments will involve a combination of two-dimensional polyacrylamide gel electrophoresis and immunochemical techniques. We furthermore propose to use the mutants to define and characterize ribosomal functional sites by a combination of biochemical, immunologic and genetic studies. This collection of well-characterized CHL cell mutants also will permit genetic experiments designed to investigate the chromosomal organization of ribosomal structural loci in tissue culture.